A bacterial NLR-related protein recognizes multiple unrelated phage triggers to sense infection.

Immune systems must rapidly sense viral infections to initiate antiviral signaling and protect the host. Bacteria encode >100 distinct viral (phage) defense systems and each has evolved to sense crucial components or activities associated with the viral lifecycle. Here we used a high-throughput AlphaFold-multimer screen to discover that a bacterial NLR-related protein directly senses multiple phage proteins, thereby limiting immune evasion.

Bacterial Shedu immune nucleases share a common enzymatic core regulated by diverse sensor domains.

Prokaryotes possess diverse anti-bacteriophage immune systems, including the single-protein Shedu nuclease. Here, we reveal the structural basis for activation of Bacillus cereus Shedu. Two cryoelectron microscopy structures of Shedu show that it switches between inactive and active states through conformational changes affecting active-site architecture, which are controlled by the protein's N-terminal domain (NTD).

Rational design yields RNA-binding zinc finger domains with altered sequence specificity.

Targeting and manipulating endogenous RNAs in a sequence-specific manner is essential for both understanding RNA biology and developing RNA-targeting therapeutics. RNA-binding zinc fingers (ZnFs) are excellent candidates as designer proteins to expand the RNA-targeting toolbox, due to their compact size and modular sequence recognition. Currently, little is known about how the sequence of RNA-binding ZnF domains governs their binding site specificity.

Loop-extrusion-mediated plasmid DNA cleavage by the bacterial SMC Wadjet complex.

Structural maintenance of chromosomes (SMC) complexes play pivotal roles in genome organization and maintenance across all domains of life. In prokaryotes, SMC-family Wadjet complexes structurally resemble the widespread MukBEF but serve a defensive role by inhibiting plasmid transformation. We previously showed that Wadjet specifically cleaves plasmid DNA; however, the molecular mechanism underlying plasmid recognition remains unclear.

Mechanistic basis for protein conjugation in a diverged bacterial ubiquitination pathway.

Ubiquitination is a fundamental and highly conserved protein post-translational modification pathway, in which ubiquitin or a ubiquitin-like protein (Ubl) is typically conjugated to a lysine side chain of a target protein. Ubiquitination is a multistep process initiated by adenylation of the Ubl C-terminus, followed by sequential formation of 2-3 Ubl~cysteine thioester intermediates with E1, E2, and E3 proteins before formation of the final Ubl-lysine isopeptide bond. Ubiquitination is conserved across eukaryotes, and recent work has also revealed at least two related bacterial pathways that perform protein conjugation in the context of antiphage immunity.

Bacterial WYL domain transcriptional repressors sense single-stranded DNA to control gene expression.

Bacteria encode a wide array of immune systems to protect themselves against ubiquitous bacteriophages and foreign DNA elements. While these systems' molecular mechanisms are becoming increasingly well known, their regulation remains poorly understood. Here, we show that an immune system-associated transcriptional repressor of the wHTH-WYL-WCX family, CapW, directly binds single-stranded DNA to sense DNA damage and activate expression of its associated immune system.

TRIM37 employs peptide motif recognition and substrate-dependent oligomerization to prevent ectopic spindle pole assembly.

Tightly controlled duplication of centrosomes, the major microtubule-organizing centers of animal cells, ensures bipolarity of the mitotic spindle and accurate chromosome segregation. The RBCC (RING-B-box-coiled coil) ubiquitin ligase TRIM37, whose loss is associated with elevated chromosome missegregation and the tumor-prone developmental human disorder Mulibrey nanism, prevents the formation of ectopic spindle poles that assemble around structured condensates containing the centrosomal protein centrobin. Here, we show that TRIM37's TRAF domain, unique in the extended TRIM family, engages peptide motifs in centrobin to suppress condensate formation.

Bacteriophage Φ21's receptor-binding protein evolves new functions through destabilizing mutations that generate non-genetic phenotypic heterogeneity.

How viruses evolve to expand their host range is a major question with implications for predicting the next pandemic. Gain-of-function experiments have revealed that host-range expansions can occur through relatively few mutations in viral receptor-binding proteins, and the search for molecular mechanisms that explain such expansions is underway. Previous research on expansions of receptor use in bacteriophage λ has shown that mutations that destabilize λ's receptor-binding protein cause it to fold into new conformations that can utilize novel receptors but have weakened thermostability.

High-sensitivity in situ capture of endogenous RNA-protein interactions in fixed cells and primary tissues.

RNA-binding proteins (RBPs) have pivotal functions in RNA metabolism, but current methods are limited in retrieving RBP-RNA interactions within endogenous biological contexts. Here, we develop INSCRIBE (IN situ Sensitive Capture of RNA-protein Interactions in Biological Environments), circumventing the challenges through in situ RNA labeling by precisely directing a purified APOBEC1-nanobody fusion to the RBP of interest. This method enables highly specific RNA-binding site identification across a diverse range of fixed biological samples such as HEK293T cells and mouse brain tissue and accurately identifies the canonical binding motifs of RBFOX2 (UGCAUG) and TDP-43 (UGUGUG) in native cellular environments.

The synaptonemal complex aligns meiotic chromosomes by wetting.

During meiosis, the parental chromosomes are drawn together to enable exchange of genetic information. Chromosomes are aligned through the assembly of a conserved interface, the synaptonemal complex, composed of a central region that forms between two parallel chromosomal backbones called axes. Here we identify the axis-central region interface in , containing a conserved positive patch on the axis component HIM-3 and the C-terminus of the central region protein SYP-5.

Alteration of Neuropilin-1 and Heparan Sulfate Interaction Impairs Murine B16 Tumor Growth.

Neuropilin-1 acts as a coreceptor with vascular endothelial growth factor receptors to facilitate binding of its ligand, vascular endothelial growth factor. Neuropilin-1 also binds to heparan sulfate, but the functional significance of this interaction has not been established. A combinatorial library screening using heparin oligosaccharides followed by molecular dynamics simulations of a heparin tetradecasaccharide suggested a highly conserved binding site composed of amino acid residues extending across the b1 and b2 domains of murine neuropilin-1.

A eukaryotic-like ubiquitination system in bacterial antiviral defence.

Ubiquitination pathways have crucial roles in protein homeostasis, signalling and innate immunity. In these pathways, an enzymatic cascade of E1, E2 and E3 proteins conjugates ubiquitin or a ubiquitin-like protein (Ubl) to target-protein lysine residues. Bacteria encode ancient relatives of E1 and Ubl proteins involved in sulfur metabolism, but these proteins do not mediate Ubl-target conjugation, leaving open the question of whether bacteria can perform ubiquitination-like protein conjugation.

An intron endonuclease facilitates interference competition between coinfecting viruses.

Introns containing homing endonucleases are widespread in nature and have long been assumed to be selfish elements that provide no benefit to the host organism. These genetic elements are common in viruses, but whether they confer a selective advantage is unclear. In this work, we studied intron-encoded homing endonuclease gp210 in bacteriophage ΦPA3 and found that it contributes to viral competition by interfering with the replication of a coinfecting phage, ΦKZ.

An essential and highly selective protein import pathway encoded by nucleus-forming phage.

Targeting proteins to specific subcellular destinations is essential in prokaryotes, eukaryotes, and the viruses that infect them. Chimalliviridae phages encapsulate their genomes in a nucleus-like replication compartment composed of the protein chimallin (ChmA) that excludes ribosomes and decouples transcription from translation. These phages selectively partition proteins between the phage nucleus and the bacterial cytoplasm.

Viral Receptor-Binding Protein Evolves New Function through Mutations That Cause Trimer Instability and Functional Heterogeneity.

When proteins evolve new activity, a concomitant decrease in stability is often observed because the mutations that confer new activity can destabilize the native fold. In the conventional model of protein evolution, reduced stability is considered a purely deleterious cost of molecular innovation because unstable proteins are prone to aggregation and are sensitive to environmental stressors. However, recent work has revealed that nonnative, often unstable protein conformations play an important role in mediating evolutionary transitions, raising the question of whether instability can itself potentiate the evolution of new activity.

Phospho-KNL-1 recognition by a TPR domain targets the BUB-1-BUB-3 complex to C. elegans kinetochores.

During mitosis, the Bub1-Bub3 complex concentrates at kinetochores, the microtubule-coupling interfaces on chromosomes, where it contributes to spindle checkpoint activation, kinetochore-spindle microtubule interactions, and protection of centromeric cohesion. Bub1 has a conserved N-terminal tetratricopeptide repeat (TPR) domain followed by a binding motif for its conserved interactor Bub3. The current model for Bub1-Bub3 localization to kinetochores is that Bub3, along with its bound motif from Bub1, recognizes phosphorylated "MELT" motifs in the kinetochore scaffold protein Knl1.

An essential and highly selective protein import pathway encoded by nucleus-forming phage.

Unlabelled: Targeting proteins to specific subcellular destinations is essential in prokaryotes, eukaryotes, and the viruses that infect them. Chimalliviridae phages encapsulate their genomes in a nucleus-like replication compartment composed of the protein chimallin (ChmA) that excludes ribosomes and decouples transcription from translation. These phages selectively partition proteins between the phage nucleus and the bacterial cytoplasm.

A phage nucleus-associated RNA-binding protein is required for jumbo phage infection.

Large-genome bacteriophages (jumbo phages) of the proposed family Chimalliviridae assemble a nucleus-like compartment bounded by a protein shell that protects the replicating phage genome from host-encoded restriction enzymes and DNA-targeting CRISPR-Cas nucleases. While the nuclear shell provides broad protection against host nucleases, it necessitates transport of mRNA out of the nucleus-like compartment for translation by host ribosomes, and transport of specific proteins into the nucleus-like compartment to support DNA replication and mRNA transcription. Here, we identify a conserved phage nuclear shell-associated protein that we term Chimallin C (ChmC), which adopts a nucleic acid-binding fold, binds RNA with high affinity in vitro, and binds phage mRNAs in infected cells.

Phosphorylation regulates tau's phase separation behavior and interactions with chromatin.

Tau is a microtubule-associated protein often found in neurofibrillary tangles (NFTs) in the brains of patients with Alzheimer's disease. Beyond this context, mounting evidence suggests that tau localizes into the nucleus, where it may play a role in DNA protection and heterochromatin regulation. The molecular mechanisms behind these observations are currently unclear.

Loop extrusion-mediated plasmid DNA cleavage by the bacterial SMC Wadjet complex.

Structural maintenance of chromosomes (SMC) protein complexes play pivotal roles in genome organization and maintenance across all domains of life. In prokaryotes, SMC family Wadjet complexes structurally resemble the widespread MukBEF genome-organizing complexes but serve a defensive role by inhibiting plasmid transformation. We previously showed that Wadjet specifically cleaves circular DNA; however, the molecular mechanism underlying DNA substrate recognition remains unclear.

Phospho-KNL-1 recognition by a TPR domain targets the BUB-1-BUB-3 complex to kinetochores.

During mitosis, the Bub1-Bub3 complex concentrates at kinetochores, the microtubule-coupling interfaces on chromosomes, where it contributes to spindle checkpoint activation, kinetochore-spindle microtubule interactions, and protection of centromeric cohesion. Bub1 has a conserved N-terminal tetratricopeptide (TPR) domain followed by a binding motif for its conserved interactor Bub3. The current model for Bub1-Bub3 localization to kinetochores is that Bub3, along with its bound motif from Bub1, recognizes phosphorylated "MELT" motifs in the kinetochore scaffold protein Knl1.

Chromatin binding by HORMAD proteins regulates meiotic recombination initiation.

The meiotic chromosome axis coordinates chromosome organization and interhomolog recombination in meiotic prophase and is essential for fertility. In S. cerevisiae, the HORMAD protein Hop1 mediates the enrichment of axis proteins at nucleosome-rich islands through a central chromatin-binding region (CBR).